The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by EXponential Enrichment,” now abandoned, U.S. Pat. No. 5,475,096 entitled “Nucleic Acid Ligands”, and U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled “Nucleic Acid Ligands” each of which is specifically incorporated by reference herein. Each of these patents and applications, collectively referred to herein as the SELEX patent applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands or aptamers, each having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX process-identified nucleic acid ligand is a specific ligand of a given target compound or molecule.
The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. The SELEX process applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX process includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
One particularly important embodiment of the SELEX process is described in U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, and U.S. patent application Ser. No. 08/443,959 filed May 18, 1995, both entitled “Photoselection of Nucleic Acid Ligands,” and both now abandoned, and U.S. Pat. Nos. 5,763,177, 6,001,577, WO 95/08003, U.S. Pat. Nos. 6,291,184, 6,458,539, and U.S. patent application Ser. No. 09/723,718, filed Nov. 28, 2000, each of which is entitled “Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,” and each of which describe a SELEX process-based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. The resulting nucleic acid ligands are referred to interchangeably as “photocrosslinking nucleic acid ligands” and “photoaptamers.” These patents and patent applications are referred to in this application collectively as “the PhotoSELEX Process Applications.” In the photoSELEX process embodiment of the SELEX process, a modified nucleotide activated by absorption of light is incorporated in place of a native base in either RNA- or in ssDNA-randomized oligonucleotide libraries. One such photoreactive nucleotide whose photochemistry is particularly well-suited for this purpose is 5-bromo-2′-deoxyuridine (5-BrdU) (Meisenheimer and Koch (1997) Crit. Rev. Biochem. Mol. Biol. 32:101-140). The 5-BrdU chromophore absorbs ultraviolet (UV) light in the 310 nm range where native chromophores of nucleic acids and proteins do not absorb or absorb very weakly. The resulting excited singlet state intersystem crosses to the lowest triplet state which specifically crosslinks with aromatic and sulfur-bearing amino acid residues of a protein target in suitable proximity (Dietz and Koch (1987) Photochem. Photobiol. 46:971-8; Dietz and Koch (1989) Photochem. Photobiol. 49:121-9; Dietz et al. (1987) J. Am. Chem. Soc. 109:1793-1797; Ito et al. (1980) J. Am. Chem. Soc. 102:7535-7541; Swanson et al. (1981) J. Am. Chem. Soc. 103:1274-1276). Crosslinking may also occur via excitation of an aromatic residue of the protein in proximity to the bromouracil chromophore (Norris et al. (1997) Photochem. Photobiol. 65:201-207). Of particular importance, excited bromouracil in DNA is relatively unreactive in the absence of a proximal, oriented, reactive amino acid (Gott et al. (1991) Biochemistry 30:6290-6295; Willis et al. (1994) Nucleic Acids Res. 22:4947-4952; Norris et al. (1997) Photochem. Photobiol. 65:201-207) or nucleotide residue (Sugiyama et al. (1990) J. Am. Chem. Soc. 112:6720-6721; Cook and Greenberg (1996) J. Am. Chem. Soc. 118:10025-10030). The importance of orientation is evident in crystal structures of protein-nucleic acid complexes which show a lock and key arrangement of the bromouracil chromophore with the aromatic amino acid residue to which it crosslinks (Horvath et al. (1998) Cell 95:963-974; Meisenheimer and Koch (1997) Crit. Rev. Biochem. Mol. Biol. 32:101-140).
In a basic embodiment, the photoSELEX process comprises the following steps:    a) A candidate mixture of nucleic acids is prepared. The candidate mixture nucleic acids comprise sequences with randomized regions including photoreactive groups, e.g. by incorporating 5-BrdU into the candidate mixture.    b) The candidate mixture is contacted with a quantity of target. Nucleic acid ligands of the target in the candidate mixture form complexes with the target;    c) The photoreactive groups in candidate nucleic acid ligands are photoactivated by irradiation. Nucleic acid ligands that have formed specific complexes with target thereby become photocrosslinked to the target;    d) Nucleic acid ligands that have become photocrosslinked to target are partitioned from other nucleic acids in the candidate mixture;    e) The nucleic acid ligands that photocrosslinked to the target are released from the target (e.g., by protease digestion if the target is a protein), and then amplified; and    f) The amplified nucleic acid ligands are used as the candidate mixture to initiate another round of the photoSELEX process.
The photoSELEX process produces nucleic acid ligands that are single- or double-stranded RNA or DNA oligonucleotides. A photoreactive group may comprise a natural nucleic acid residue with a relatively simple modification that confers increased reactivity or photoreactivity to the nucleic acid residue. Such modifications include, but are not limited to, modifications at cytosine exocyclic amines, substitution with halogenated groups, e.g., 5′-bromo- or 5′-iodo-uracil, modification at the 2′-position, e.g., 2′-amino (2′-NH2) and 2′-fluoro (2′-F), backbone modifications, methylations, unusual base-pairing combinations and the like. For example, photocrosslinking nucleic acid ligands produced by the photoSELEX process can include a photoreactive group selected from the following: 5-bromouracil (BrU), 5-iodouracil (IU), 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-azidoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuridine, 8-bromo-2′-deoxyadenine, 5-iodo-2′-deoxyuracil, 5-iodo-2′-deoxycytosine, 5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil, 7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine, and 7-deaza-7-bromoguanine. Preferably, the photoreactive group will absorb light in a spectrum of the wavelength that is not absorbed by the target or the non-modified portions of the oligonucleotide. In preferred embodiments of the photoSELEX process, the photoreactive nucleotides incorporated into the photocrosslinking nucleic acid ligands are 5-bromo-2′-deoxyuridine (5-BrdU) and 5-iodo-2′-deoxyuridine (5-IdU). These nucleotides can be incorporated into DNA in place of thymidine nucleotides.
Photocrosslinking nucleic acid ligands produced by the photoSELEX process have particular utility in diagnostic or prognostic medical assays. In one such embodiment, photocrosslinking nucleic acid ligands of targets implicated in disease are attached to a planar solid support in an array format, and the solid support is then contacted with a biological fluid to be analyzed for the presence or absence of the targets. The photocrosslinking nucleic acid ligands are photoactivated and the solid support is washed under very stringent, aggressive conditions (preferably under conditions that denature nucleic acids and/or proteins) in order to remove all non-specifically bound molecules. Bound target is not removed because it is covalently crosslinked to nucleic acid ligand via the photoreactive group. The ability to photocrosslink, followed by stringent washing, allows diagnostic and prognostic assays of unparalleled sensitivity and specificity to be performed. Arrays (also commonly referred to as “biochips” or “microarrays”) of nucleic acid ligands, including photocrosslinking nucleic acid ligands and aptamers, and methods for their manufacture and use, are described in U.S. Pat. Nos. 6,242,246, U.S. patent application Ser. No. 08/211,680, filed Dec. 14, 1998, now abandoned, WO 99/31275, U.S. patent application Ser. No. 09/581,465, filed Jun. 12, 2000, U.S. Pat. Nos. 6,503,715, and 6,458,543, each of which is entitled “Nucleic Acid Ligand Diagnostic Biochip.” These patents and patent applications are referred to collectively as “the biochip applications,” and are each specifically incorporated herein by reference in their entirety.
Automated methods and apparatus for the generation of photocrosslinking nucleic acid ligands are provided in U.S. patent application Ser. No. 09/993,294, filed Nov. 21, 2001, U.S. patent application Ser. No. 09/815,171, filed Mar. 22, 2001, U.S. patent application Ser. No. 09/616,284, filed Jul. 14, 2000, U.S. patent application Ser. No. 09/356,233, filed Jul. 16, 1999, U.S. patent application Ser. No. 09/232,946, filed Jan. 19, 1999, each of which is entitled “Method and Apparatus for the Automated Generation of Nucleic Acid Ligands.” Given the rapidity with which these highly parallel, automated methods can generate photocrosslinking nucleic acid ligands, it is desirable to have multiplexed methods for evaluation of the specificity and dose-response characteristics of those photocrosslinking nucleic acid ligands. The present invention includes such methods.